Structural approaches to understand the promiscuous substrate specificity of Mesh hydrolase. We wish to test new regulators in E. coli and need to modify Mesh hydrolase so it degrades (p)ppApp but not (p)ppGpp. Relseq is an enzyme that degrades (p)ppGpp but not (p)ppApp.. The crystal structures of both fly, human Mesh and bacterial RelSeq hydrolases are available together with abundant sequence data. Tamara James used this information to model differences between the catalytic pocket of each enzymes to predict residues that could discriminate between the adenine or guanine purine rings of (p)pp(A,G)pp. Missense mutants of Mesh were constructed, overexpressed, purified and their specific activities towards ppApp and ppGpp measured qualitatively by thin layer chromatography (Tamara James and Nathan Thomas). Two rounds of analysis now have been completed. Separate regions of the catalytic pocket are now defined by two mutations each as either guanine-specific or adenine-specific. Future experiments will provide quantitative measurements of catalytic constants for both classes of substrates. Qualitatively, it is already evident that mutant Mesh is not yet completely (p)ppApp specific and more mutations are needed to achieve the very strong differences is Mesh specificities. This work is also a collaboration with Dr. K. Potrykus, who discovered that Mesh degrades (p)ppApp. Genetic approaches suggest Mesh uniquely hydrolyzes an unknown substrate in E. coli essential for growth. A Mesh gene cloned in an inducible plasmid allows asking if it possesses a growth altering activity that differs from those of bacterial hydrolases known to degrade only (p)ppGpp but not (p)ppApp. This general approach is powerful because it does not require knowing the full range of possible substrates of both hydrolases. Mesh clones are not viable in hosts completely lacking (p)ppGpp. When Mesh is the sole source of hydrolase in strains with ppGpp at levels high enough to themselves inhibit growth, normal growth occurs indicating that Mesh hydrolase activity has two effects:1) ppGpp is degraded to restore rapid growth otherwise lost due to ppGpp and 2) the toxicity of Mesh is reversed presumably because hydrolysis of an essential substrate is reduced by ppGpp hydrolysis. This essential substrate is predicted to be a purine ribonucleotide with pyrophosphate esterified to a ribosyl 3-hydroxyl or a compound with this residue. Future experiments will be aimed at determining the identity of this Mesh substrate. Tests of Mesh mutants with modified substrate specificity will be important to this search. Studies of regulation by pGpp vs ppGpp or pppGpp. We have contributed to characterizing a new regulator (pGpp) made by RelQ in Gram positive bacteria in the lab of Dr. J. Lemos. This is an important collaboration because it opens the search for regulation specific to pGpp that is not shared by (p)ppGpp. So far in E. coli, we find potent regulation by ppGpp is invariably shared to a lesser extent by pppGpp 1. Lemos and co-workers have constructed Enterococcus faecalis mutants in which RelQ is the only synthetase and these accumulate pGpp and (p)ppGpp. Other mutants lack RelQ and accumulate only (p)ppGpp. The absence of pGpp has been shown to weaken starvation responses and pathogenicity but the specific mechanisms are so far elusive. We compared the inhibitory activity of pGpp to (p)ppGpp with respect to their ability to inhibit initiation of E. coli ribosomal RNA transcription. Only a very weak inhibitory activity was found for pGpp, which ranks at the bottom of the potency hierarchy: ppGpp>pppGpp>>pGpp. So far, all regulatory activities found for pGpp are shared to some extent with ppGpp or pppGpp. This is not unexpected, since searches have explored only examples where (p)ppGpp is active. (p)ppGpp regulation affects chaperone phenotypes and chaperone activities are probably shared among RNAP accessory proteins (dksA greA, greB and traR). We have applied genetics to resolve a dilemma raised by finding (p)ppGpp binds to a RNAP site that includes portions of the omega subunit (1). The dilemma arises because omega deleted cells do not have a phenotype, which is inconsistent with the many phenotypes of (p)ppGpp0 strains. Our omega deleted strains are noted by others to have immature RNAP subunits bound to the GroEL chaperone and as well as folding difficulties during in vitro assembly. We wondered if RNAP maturation is important enough that other chaperones might completely take over when omega is missing. If so, deletion of chaperone genes might unmask a growth dependence on omega. We now have supporting evidence. Overall there are many functional overlaps between omega, chaperones, RNAP accessory proteins and (p)ppGpp. The DksA protein, which can act in concert with (p)ppGpp regulation is named after its ability to reverse a phenotype due to a dnaK chaperone deletion (DNA K suppressor). We created and studied a matrix of all the possible combinations (of single, double and triple) deletions of omega, relA (ppGpp), and each of four chaperones: dnaK, dnaJ, tig, or clpB. The double mutants quickly led to the discovery that the relA/dnaK (or relA/dnaJ) combination lowered survival temperatures from 370 to 240 omega. This appears to be a striking new phenotype for relA. This associates (p)ppGpp with dnaK/J and as just mentioned, dnaK/J are also linked to dksA by suppression. The next discovery required triple mutants: cells deleted for omega failed to grow at 320 but + omega grew at high temperatures, 420. All double mutant combinations were insensitive. This finding links omega with (p)ppGpp, tig, clpB and indirectly with GroE. DksA function was linked to the triple mutant phenotype by showing that excess DksA reverses the triple mutant phenotype. We reported (2) that excess of DksA or GreA could reverse some phenotypes of (p)ppGpp0 mutants and could themselves function as synthetic lethals. This implies a GreA functional redundancy with DksA. The question arises as to what sorts of functions are shared by these secondary channel RNAP associated proteins. A few years ago in our lab R. Harinarayanan found overproduced GreA or GreB reversed the temperature sensitivity of dnaKJ mutants similar to overproduced DksA. This implies chaperone activity for GreA and GreB, usually known as able to reverse arrested transcription complexes. Mutant variants of GreA and GreB, designated GreA*, GreB* lack transcription activities are but are still able to reverse the temperature sensitivity of dnaKJ mutants; DksA* behaves similarly. This activity of DksA* extends to reversing the temperature sensitivity of the tig/clpB, (p)ppGpp and omega triple mutant. The chaperone-like activities shared by these proteins, together with their involvement in regulation by (p)ppGpp is a new common feature. This seems to be their structure, which uniformly consists of a coiled-coil topped by a larger globular domain. The question now becomes how this structure could have chaperone activity. There is literature evidence that GreA protein does have chaperone activity. This question will be studied more closely during the coming year as well as the role of omega in (p)ppGpp regulation.